Genetic effect of an A/G polymorphism in the HSP70 gene on thermotolerance in chicken.

Studying thermotolerance is important for the prevention of thermostress in chickens. This study aimed to analyze the effect of mutations in the heat shock protein 70 (HSP70) gene on chicken thermotolerance. The C.-69A>G SNP in the 5'-flanking region of the HSP70 gene was genotyped in Lingshan and White Recessive Rock (WRR) chickens. Association of this SNP with thermotolerance traits revealed it to be significantly associated with CD4+/CD8+, and potentially associated with heterophil-to-lymphocyte ratio in WRR chickens exposed to thermoneutral temperature (15°C). Online prediction detected a putative myeloid zinc finger protein 1 binding factor in the C.-69A>G mutation. Under acute thermostress, mRNA levels of HSP70 in individuals with different C.-69A>G genotypes varied in the heart, leg muscle, and liver tissues. The HSP70 protein was expressed at higher levels in individuals with the GG genotype than in those with the AA genotype. In heart and liver, protein expression of HSP70 in individuals with the GG genotype was significantly higher than in those with the AA genotype. In leg muscle, protein expression was higher in birds with the GG genotype than in those with the AA and AG genotypes. Luciferase activity of the GG genotype was significantly higher than that of the AA genotype, suggesting that the C.-69A>G SNP regulates HSP70 gene expression. These results indicate that the C.-69A>G SNP in the 5'-flanking region of the HSP70 gene might affect chicken thermotolerance and that the GG genotype might be advantageous for the prevention of thermostress.

Characterization of HSP70 and its expression in tissue: correlation with physiological and immune indices in goose (Anser cygnoides) serum.

We cloned the goose heat shock protein 70 gene (HSP70), to determine its sequence variation and elucidate its mRNA expression. We designed primers to amplify the entire goose HSP70 sequence. We used 10 commercial Wuzong goslings in a heat-stress experiment. We collected tissue samples for RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR). We analyzed the variation in expression of goose HSP70 before and after heat stress. We constructed a DNA pool from six different species, for single nucleotide polymorphism (SNP) screening. We detected 18 SNPs and selected three of these SNPs for correlation analysis with biological and immune traits in 200 Wuzong geese. We showed that T+237C was significantly correlated with the serum corticosterone level, whereas T+1122C was significantly correlated with the heterophil to lymphocyte ratio. Goose HSP70 contained no introns. The results of qRT-PCR analysis revealed significant gender differences in the expression of goose HSP70 at 40°C but not at 25°C; moreover, in general, expression was significantly higher at 40°C than at 25°C. With the exception of the leg muscle and cerebellum, HSP70 expression was significantly higher in male geese than in female geese. Our results indicate that goose HSP70 plays an important role in response to severe heat stress.

Analysis of genetic diversity of the heat shock protein 70 gene on the basis of abundant sequence polymorphisms in chicken breeds.

This study was designed to detect the sequence variation of the chicken heat shock protein 70 (HSP70) gene. A total of 102 individuals from 8 native Chinese breeds together with Dwarf White Chicken and Red Junglefowl were used to detect sequence variations. The coding regions of the chicken HSP70 gene from 102 individuals were cloned and sequenced. Thirty-six variations were identified, which included 34 single nucleotide polymorphisms and 2 indel mutations. Fifty-seven haplotypes were observed, of which, 43 were breed-specific and 14 were shared. There were 7 Red Junglefowl-specific haplotypes, while Haidong and Silkie only had 2 specific haplotypes. Eleven and 3 haplotypes were shared between and within species, respectively. The variation in nucleotide diversity (Pi) and average number of nucleotide differences (K) among species were consistent. The total Pi of HSP70 was 0.0016, and the total K was 4.1998. The Pi value of Red Junglefowl was the highest (0.0018) and K was 4.8000, while the Pi of Silkie was the lowest (0.0010) and K was 2.5000. These results demonstrated that variation in chicken HSP70 was abundant between and within species.

Promoter methylation negatively correlated with mRNA expression but not tissue differential expression after heat stress.

DNA methylation plays a central role in gene expression. In this study, we detected the promoter methylation pattern of the chicken heat shock protein 70 (HSP70) gene and its association with messenger RNA (mRNA) expression before and after heat shock. The results showed that mRNA expression increased in response to heat stress and peaked at 3 h before dropping. Hypomethylation of the HSP70 promoter occurred in all of the groups studied, but the difference between groups within tissue type was not significant. The DNA methylation level of the control and the 6-h treatment groups was slightly higher than that of the 3-h treatment group in brain tissue and leg muscle. Correlation analysis between mRNA expression and DNA methylation of HSP70 showed that DNA methylation was negatively associated with mRNA expression in leg muscle (P = 0.0124), indicating that DNA methylation may be negatively associated with the expression of HSP70, although the difference was not significant. We concluded that the expression of HSP70 is heat inducible and tissue dependent and that heat induction may correlate with DNA methylation pattern in the HSP70 promoter, whereas tissue dependence is unrelated to DNA methylation pattern.

Bulk-Fill Composites: Effectiveness of Cure With Poly- and Monowave Curing Lights and Modes.

This study compared the effectiveness of cure of bulk-fill composites using polywave light-emitting diode (LED; with various curing modes), monowave LED, and conventional halogen curing lights. The bulk-fill composites evaluated were Tetric N-Ceram bulk-fill (TNC), which contained a novel germanium photoinitiator (Ivocerin), and Smart Dentin Replacement (SDR). The composites were placed into black polyvinyl molds with cylindrical recesses of 4-mm height and 3-mm diameter and photopolymerized as follows: Bluephase N Polywave High (NH), 1200 mW/cm2 (10 seconds); Bluephase N Polywave Low (NL), 650 mW/cm2 (18.5 seconds); Bluephase N Polywave soft-start (NS), 0-650 mW/cm2 (5 seconds) → 1200 mW/cm2 (10 seconds); Bluephase N Monowave (NM), 800 mW/cm2 (15 seconds); QHL75 (QH), 550 mW/cm2 (21.8 seconds). Total energy output was fixed at 12,000 mJ/cm2 for all lights/modes, with the exception of NS. The cured specimens were stored in a light-proof container at 37°C for 24 hours, and hardness (Knoop Hardness Number) of the top and bottom surfaces of the specimens was determined using a Knoop microhardness tester (n=6). Hardness data and bottom-to-top hardness ratios were subjected to statistical analysis using one-way analysis of variance/Scheffe's post hoc test at a significance level of 0.05. Hardness ratios ranged from 38.43% ± 5.19% to 49.25% ± 6.38% for TNC and 50.67% ± 1.54% to 67.62% ± 6.96% for SDR. For both bulk-fill composites, the highest hardness ratios were obtained with NM and lowest hardness ratios with NL. While no significant difference in hardness ratios was observed between curing lights/modes for TNC, the hardness ratio obtained with NM was significantly higher than the hardness ratio obtained for NL for SDR.

Copy number variation identification and analysis of the chicken genome using a 60K SNP BeadChip.

Copy number variation (CNV) is an important source of genetic variation in organisms and a main factor that affects phenotypic variation. A comprehensive study of chicken CNV can provide valuable information on genetic diversity and facilitate future analyses of associations between CNV and economically important traits in chickens. In the present study, an F2 full-sib chicken population (554 individuals), established from a cross between Xinghua and White Recessive Rock chickens, was used to explore CNV in the chicken genome. Genotyping was performed using a chicken 60K SNP BeadChip. A total of 1,875 CNV were detected with the PennCNV algorithm, and the average number of CNV was 3.42 per individual. The CNV were distributed across 383 independent CNV regions (CNVR) and covered 41 megabases (3.97%) of the chicken genome. Seven CNVR in 108 individuals were validated by quantitative real-time PCR, and 81 of these individuals (75%) also were detected with the PennCNV algorithm. In total, 274 CNVR (71.54%) identified in the current study were previously reported. Of these, 147 (38.38%) were reported in at least 2 studies. Additionally, 109 of the CNVR (28.46%) discovered here are novel. A total of 709 genes within or overlapping with the CNVR was retrieved. Out of the 2,742 quantitative trait loci (QTL) collected in the chicken QTL database, 43 QTL had confidence intervals overlapping with the CNVR, and 32 CNVR encompassed one or more functional genes. The functional genes located in the CNVR are likely to be the QTG that are associated with underlying economic traits. This study considerably expands our insight into the structural variation in the genome of chickens and provides an important resource for genomic variation, especially for genomic structural variation related to economic traits in chickens.